摘要:

ABSTRACTThe multitude of G-protein coupled receptor (GPR) superfamily cDNAs recently isolated has exceeded the number of receptor subtypes anticipated by pharmacological studies. Analysis of the sequence similarities and unique features of the members of this family is valuable for designing strategies to isolate related cDNAs, for developing hypotheses concerning substrate-ligand and receptor-effector interactions, and for undertanding the evolution of these genes. We have compiled and aligned the 74 unique amino acid sequences published to date and review the present understanding of the structural motifs contributing to ligand binding and G-protein coupling.

ISSN:1044-5498    

DOI:10.1089/dna.1992.11.1

年代:1992

数据来源: MAL

摘要:

ABSTRACTHuman cDNAs encoding the precursor to pituitary adenylate cyclase activating polypeptide (PACAP) were cloned from human testis and cerebral cortex cDNA libraries. Nucleotide sequencing revealed that cDNA from the testis library encoded the entire precursor for PACAP, while cDNA from the brain library represented only the carboxy-terminal half of the precursor. The predicted human PACAP precursor consisted of 176 amino acid residues and was very similar to the ovine one (82%). Both human and ovine precursors contained both PACAP and another peptide, PACAP-related peptide (PRP), having 29 amino acids. PACAP and PRP were preceded and followed by paired basic amino acids, recognized as important for post-translational processing. The PACAP precursor resembles the vasoactive intestinal peptide (VIP) precursor, which contains VIP and peptide histidine methionine/isoleucine amide (PHM/PHI). Structurally, PRP had some similarity to PHM/PHI, growth hormone-releasing hormone (GRH) and PACAP. Northern blot analysis indicated that a 3.0-kb transcript was expressed in the ovine hypothalamus. Tissue distribution of PACAP mRNA was also clarified in the rat. Southern blot analysis of human genomic DNA gives single bands with six restriction enzymes, indicating that a single copy of the PACAP gene is contained in a haploid genome. The cDNA for human PACAP precursor was expressed using COS-7 and Chinese hamster ovary (CHO) cells. Immunoreactive PACAP was secreted into the culture media of both transfected cell lines.

ISSN:1044-5498    

DOI:10.1089/dna.1992.11.21

年代:1992

数据来源: MAL

摘要:

ABSTRACTThe rat intestinal fatty acid binding protein (I-FABP) gene has been used as a model to study temporal and spatial differentiation of the gut epithelium while its protein product has been used as a model for examining the atomic details of noncovalent fatty acid-protein interactions. We have isolated the mouse I-FABP gene (Fabpi) and determined its nucleotide sequence. Comparisons of the orthologous mouse, rat, and human I-FABP genes revealed three conserved domains in their otherwise divergent 5′ nontranscribed sequences. RNA blot hybridization and multilabel immunocytochemical methods were used to compare the developmental stage-specific patterns of activation of the rat and mouse genes. In addition,Fabpiexpression in enterocytes was examined as a function of their differentiation along the crypto-to-villus and duodenal-to-colonic axes of the small intestine. Based on the similar temporal and geographic patterns of mouse and rat I-FABP expression described here and the results of our earlier studies of transgenic mice containing ratFabpi/human growth hormone fusion genes, we propose that one of the conserved domains, spanning nucleotides −500 to −419 in mouseFabpi, and/or a 14-bp element, are necessary for establishing and maintaining its region-specific expression along the duodenal-to-colonic axis of the perpetually renewing gut epithelium. Finally, predictions of the structure of mouse I-FABP using the refined 2.0 Å model of rat I-FABP, suggest that a proline found at position 69 of the mouse, but not rat, protein may affect its ligand binding prop

ISSN:1044-5498    

DOI:10.1089/dna.1992.11.31

年代:1992

数据来源: MAL

摘要:

ABSTRACTThe insulin-like growth factor-I receptor (IGFIR) is a membrane-bound glycoprotein that mediates the action of insulin-like growth factors. The cDNAs for the human IGFIR have been cloned and expressed, but the structures of the gene and its promoter have not been elucidated. In this study, we isolated an IGFIR promoter clone from a human chromosome 15 library. This clone contained the promoter, first exon, and a portion of the first intron. Sequence analysis of the 5′ region that contained the promoter revealed that it lacked both TATA and CAAT boxes. The promoter contained binding sites for the transcription factors Sp1, AP-2, and the epidermal growth factor receptor transcription factor (ETF). Primer extension analysis of IGFIR mRNA indicated the presence of a single transcription start site 1,012 bp upstream from the ATG. When the putative promoter was ligated into a promoterless CAT vector and transfected into HEPG2 cells, CAT activity was expressed, indicating that promoter activity was contained in this fragment. Other constructs containing the promoter and portions of the 5′ untranslated region were used in transfection studies, and indicated that the 5′ untranslated regions may play a role in promoter activity. Comparison of the human IGFIR promoter with that of the rat IGFIR promoter revealed significant sequence homology. Comparison of the IGFIR promoter with that of the human insulin receptor (IR) revealed structural similarities, although the arrangement of promoter elements dif

ISSN:1044-5498    

DOI:10.1089/dna.1992.11.43

年代:1992

数据来源: MAL

摘要:

ABSTRACTWe have determined the primary structure of a 4.7-kb portion of the ribosomal DNA intergenic spacer from cultured cells of the mosquito,Aedes albopictus. Immediately upstream from the 18S rRNA gene was a 753-bp sequence containing two regions similar to known RNA polymerase I promoters, each preceded by potential transcription termination signals. Upstream from this putative promoter region was a 3.15-kb tandem array of 17 direct repeats with a consensus sequence length of 201 bp. The 201-bp repeats contained imperfect antisense duplications of 11-bp core domain regions in the putative RNA polymerase I promoters, and sequences of possible significance in recombination. Farthest upstream of the 18S rRNA gene was an 803-bp region containing two copies each of 34-, 48-, and 64-bp elements separated by apparently unique sequence. This first detailed structural analysis of a ribosomal DNA intergenic spacer from a member of the lower Diptera has revealed features similar to those described for the higher Diptera as well as conserved motifs presumably critical to rRNA transcription.

ISSN:1044-5498    

DOI:10.1089/dna.1992.11.51

年代:1992

数据来源: MAL

摘要:

ABSTRACTThe mammalian DNA β-polymerase (β-pol) gene is constitutively expressed in cultured cells as a function of growth stage and DNA replication, but is expressed in rodents in a tissue-specific fashion. As revealed by transient expression experiments with wild-type and mutated β-pol promoter fusion genes, the cloned human β-pol promoter is transcriptionally regulated by signals acting through the single palindromic sequence (GTGACGTCAC) known as an ATF/CRE-binding site centered at position −45 in the core promoter. Although the mere presence of the ATF/CRE palindromic sequence in a promoter does not always confer cAMP responsiveness or protein binding over and around the ATF/CRE sequence, we find that agents that increase cAMP levels (forskolin and IBMX) in HeLa cells activate the β-pol promoter; activation also can be observed by coexpression of the protein kinase A catalytic subunit. Experiments with mutagenized β-pol promoters indicate that the ATF/CRE-binding site mediates these effects. Thus, the ATF/CRE-binding site in the context of this TATA-less constitutive promoter is able to respond to the kinase A signal transduction

ISSN:1044-5498    

DOI:10.1089/dna.1992.11.61

年代:1992

数据来源: MAL

摘要:

ABSTRACTA gene (eft-1) encoding an elongation factor 2-like protein was isolated from a region adjacent to the polyubiquitin gene,ubq-1, ofCaenorhabditis elegans. Sequence analysis of genomic and cDNA clones revealed that the deduced amino acid sequence of the protein (EFT-1) is 38% identical to that of mammalian andDrosophilaelongation factor 2 (EF-2). The entireeft-1gene is approximately 3.8 kb in length and contains 5 exons separated by short introns of 46–75 bp. The 2,547-bp open reading frame predicts a protein of 849 amino acid residues (calculated Mr, 96,151). Conserved sequences shared among a variety of GTP-binding proteins including EF-2 are found in the amino-terminal third of EFT-1. The carboxy-terminal half contains regions with 40–57% similarity (including conservative changes) with segments characteristic of EF-2 and its prokaryotic homolog, EF-G. However, the histidyl residue target for ADP-ribosylation of EF-2 by diphtheria toxin is replaced by tyrosine in EFT-1. Southern and Northern blot analyses indicate thateft-1is a single-copy gene that is expressed at all stages of nematode development. Amplification of fragments encoding highly conserved regions of EF-2 using the polymerase chain reaction led to the isolation of a fragment encoding the modifiable histidyl residue and which likely represents part of theC. elegansEF-2 gene (eft-2). This suggests that EFT-1 is not theC. eleganshomolog of EF-2, but a closely related prot

ISSN:1044-5498    

DOI:10.1089/dna.1992.11.71

年代:1992

数据来源: MAL

摘要:

ABSTRACTWe have developed an improved method for determining CAT activity directed by stably (transgenic mice) or transiently (tissue culture cell lines) introduced CAT reporter gene constructs. The procedure is based on the use of a new buffer system which considerably increases the stability of the CAT enzyme during the preparation of the crude cell extracts. When compared to other procedures, our method enables an increase of up to 100-fold in the sensitivity of the assay, depending on the transgenic tissue tested. Furthermore, a strong increase (up to 23-fold) was also observed with various promoter/CAT constructs transiently transfected in established tissue culture cell lines. This increase in sensitivity provides a significant reduction in the time required to perform the CAT assay when strong promoters are studied (from 18 to 1 hr) and is also very useful for the analysis of CAT gene expression driven by weak promoters.

ISSN:1044-5498    

DOI:10.1089/dna.1992.11.83

年代:1992

数据来源: MAL